Organic peptide containing substance recovered from blood having insulin-like activity

ABSTRACT

Organic peptide containing substances with insulin-like activity derived from blood or a blood component of a warm-blooded animal, which comprises the steps of pre-treating the blood or blood component by adjusting the pH thereof to a value of from 2 to 4 or of from 8 to 10, incubating the pre-treated blood at a temperature of from about 15 to about 40 DEG  C for a period of at least four hours, cooling the incubated blood to a temperature of less than 10 DEG  C, and recovering from the treated blood after neutralization organic peptide substances having a molecular weight of from about 1000 to about 5000, and an insulin-like activity of from about 14 to about 40 mu U/mg solid.

PRIOR APPLICATION

This application is a division of copending, commonly assigned U.S.patent application Ser. No. 748,956 filed Dec. 9, 1976, now U.S. Pat.No. 4,085,204.

This invention relates to a process for obtaining insulin-like activesubstances and to substances obtained by such process. Moreparticularly, the invention relates to a process for recoveringinsulin-like active substances from one or more blood components of awarm blooded animal.

It is known that certain substances recovered from the blood of warmblooded animals possess a certain amount of insulin-like activity alongwith other activities such as respiration stimulating activity andgrowth regulating activity. Such an insulin-like activity is for exampleposessed by the products described in Arz. Forsch. 18, 1019 (1968) aswell as in J. Cell. Physiol. 79, 319 (1972) which are products isolatedby special procedures from the blood of warm blooded animals, preferablyfrom blood or serum of calves.

It is an object of this invention to provide a process specificallydirected to obtaining substances having insulin-like activity, orsubstances having a high proportion of insulin-like active compounds.Said insulin like active substances are, in accordance with theinvention, recovered from blood components derived from warm bloodedanimals other than human, which is of considerable advantage both froman efficacy point of view and furthermore from a costs point of view.Thus, the insulin-like active substances isolated from the blood of warmblooded animals, preferably from one or more blood components derivedfrom sexually immature calves, possess a quantitatively higherproportion of insulin-like active substances which activity is notinhibited by insulin antibodies present either in the human or animalsystem.

Whilst blood or serum having insulin-like activity only posesses anactivity of 2.5 to 8 μU/mg solids content, it has now been found that bya particular treatment of blood or a blood component, a product can berecovered which has a high proportion of substances having insulin-likeactivity, namely an activity of from about 14 to about 40 μU/mg solidscontent.

The process in accordance with the invention for obtaining insulin-likeactive substances comprises the steps of pre-treating blood or a bloodcomponent by adjusting the pH thereof to a value of from 2 to 4,incubating the pre-treated blood at a temperature of from about 16° toabout 40° C. for a period of a least four hours, cooling the incubatedblood to a temperature of less than 10° C., and after neutralisation,recovering from the treated blood conponents organo-peptide containinginsulin-like active substances having a molecular weight of from about350 to 5000 and an insulin-like activity of from about 14 to about 40μU/mg solid.

The process described above can be equally well effected by adjustingthe pH of the blood or blood component to a value of from 8 to 10 andthen following the procedure described above.

Prior to adjustment of the pH, the blood or blood component should bediluted with from about one half to about four volumes of a hydrophilicdiluent.

The blood or blood component employed is normally whole blood, plasma,serum, erythrocyte suspension or thrombocyte suspension. However, theblood component may constitute the residue remaining after inorganic ororganic substances of molecular weight less than about 5000 have beensubstantially removed from the blood or blood component. Similarly, theblood component may comprise the residue remaining after inorganic ororganic substances of molecular weight less than about 1000 have beenremoved from the blood or blood component.

The procedure by which the insulin-like active substances may berecovered from the treated blood or blood component may preferablycomprise dialysing such treated blood or blood component against amembrane adapted to pass molecules having a molecular weight of lessthan about 6,000, and recovering the insulin-like active substances fromthe dialysate.

The insulin-like active substances recovered by the process describedabove may be subjected to an insulin-like active enrichment process, inwhich inorganic salts and lower molecular weight substances are removedfrom the recovered insulin-like active substance to obtain aninsulin-like active product having an insulin-like activity of fromabout 35 μU/mg to about 200 μU/mg solid.

It is recommended that the recovered insulin-like active substances orinsulin-like active product be concentrated down or diluted to obtain anisotonic solution having a solids content of about 2.5 to 20 mg/ml withan insulin-like activity of about 500 μU/mg, where necessary byadjustment with glucose or sodium chloride.

The insulin-like active organo-peptide containing substances obtainedfrom blood or a blood component of a warm-blooded animal have amolecular weight of from about 350 to 6000, a non-suppressibleinsulin-like activity in the rat adipose tissue test of from about 35 to200 μU/mg solids, solubility in ethanol, stability to heat at neutralpH, a positive ninhydrin and amido black G-reaction, a UV maximum at 258millimicrons indicating presence of purine derivatives, an amino acidcontent after hydrolysis providing a peptide content of from 25 to 30%of the dry weight of the salt free substances, with the following aminoacids: Glutamic acid, Leucine, Glycine, Serine, Asparginic acid,Alanine, Isoleucine, Histidine, Valine, Threonine, Lysine, Methionine,Thyrosine, Proline and Arginine, an insulin-like activity indicated byuptake of glucose in adipose tissue, a blood sugar lowering effect inwarm-blooded animals and an inhibition of lipolysis and the growthincrease of cell cultures such as fibroblasts.

The insulin-like activity of the substances obtained by the process ofthe invention is determined using adipose tissue of rats in accordancewith the method described in J. Clin. Invest. 39, 1487-1498 (1969). Foreach adipose tissue test, which consists of twenty-four individualtests, three male rats of a weight of about 180 to 200 g are employed.After preparation of the two epidymal adipose tissues, these are cut andso divided that each of 24 reaction flasks contains a forward, middle orrearward section of the adipose tissue (totalling 80-100 mg adiposetissue). Each determination is carried out in triplicate, in which 35 mgportions of the preparations on which insulin-like activity is to bedetermined is weighed out each time and dissolved in 7 ml ofKrebs-Ringer bicarbonate buffer which contains 100 mg% gelatine and 250mg% glucose. The insulin curve employed for guaging insulin-likeactivity covers a range of from 63 to 252 μU of insulin. The completecharges contain about 100 mg of adipose tissue, 9.5 mg of substance onwhich the insulin-like activity is to be determined (or also standardcontrols or different insulin concentrations), 1.9 ml Krebs-Ringerbicarbonate buffer and 0.1 ml glucose --1--C¹⁴. The charges areincubated at 37° C. for 2 hours.

An amount of 0.4 ml hyamine is then injected into the reaction flask forthe purpose of collecting C¹⁴ O₂ and the while maintaining a closedsystem 0.25 ml of a 2N sulpluric acid solution is introduced with theaid of a cannula to liberate the C¹⁴ O₂. After half an hour incubationof the individual reaction flasks at a temperature of 37° C., thehyamine is transferred from the absorption-acting synthetic materialcaps into synthetic material flasks, which serve as counter flasks andwhich are filled with 8 ml toluene scintillation solution. Thescintillation solution employed consists of 4 g of 2,5-diphenyl-oxazole,100 mg 1,4-bis-2-(4-methyl-5-phenyl-oxazolyl) benzene and one liter oftoluene. The radioactivity is measured in a liquid scintillationspectro-meter.

When the same test is carried out with the addition of insulinantibodies, there is no lowering or only a slight lowering of themeasured values.

The determination of a stimulation or inhibition of lipolysis in adiposetissue is carried out by employing adipose tissue in which a lipolysisis induced with 0.4 μg adrenaline/ml or 0.1 μg/ml of glucagon. Theconcentration changes of the free fatty acids and glycerine is thendetermined in the medium before and after incubation with variousconcentrations of the substance whose insulin-like activity is to betested. An insulin comparison curve is produced with insulin solutionsof different concentrations between 1 and 100 μU/ml. The test methodemployed is described in detail in Arz. Forsch. 18, 1019 (1968).

The activity of the substances produced in accordance with the inventionon the blood sugar level is also obtained in in vivo tests. In thistest, Wistar rats having a weight of 250 g are injected intravenouslywith 3 g of glucose/kg. A comparison group, at the same time as theglucose injection, receives 2 mg of the substance of the inventionobtained in the Example 4 (15 to 20 μU/mg). The blood sugar level in thecontrol animals increases within 15 minutes up to 400 mg% and normalisesagain after 60 minutes to about 120 mg%. In the test animals treatedwith the active substances of the invention, a 20% lower blood sugarlevel is observed in the same time period.

Injection of the same substance of the invention in Alloxan-diabeticrats leads to a statistically significant blood sugar decrease incomparison with Alloxan-diabetic animals injected intravenously with thesame amount of physiological salt solution in place of the substance ofthe invention.

The insulin-like active substances obtained in accordance with theprocess of the invention are useful blood sugar lowering agents in warmblooded animals in hyperglycemic condition, as for example specificallyindicated by results obtained in the in vivo tests carried out onhyperglycemic rats described above. For this use, satisfactory resultsare obtained at daily dosages of from about 200 μU to about 500 μU/kganimal body weight, conveniently administered in divided doses two tothree times a day. The preferred means of administration is byintravenous infusion, and the product of the invention is accordinglymost conveniently provided in injectable liquid form.

A specific advantageous use of the insulin-like active products of theinvention is as a supplement to regular insulin treatment of diabeticpatients exhibiting the common ulceration side effects following on longterm treatment with insulin. The advantage of said supplementarytreatment is firstly attributed to the greater proportions ofinsulin-like active substances which are not suppressible by insulinantibodies, present in the substances recovered by the process inaccordance with the invention, and secondly by virtue of growthregulating properties promoting healing which are present in saidsubstances.

The influence of the pH value on the amount of insulin-like activesubstance obtained can be seen from the following Table. It will benoted that at the pH ranges of 2 to 4 or 8 to 10 chosen in the processof the invention as against other pH values, a particularly high totalyield of substances having insulin-like activity is obtained.

                  Table                                                           ______________________________________                                        Influence of pH value on the recovery of substances having -insulin-like      activity                                                                                 Yield in g  Insulin-like                                                                            Total                                                   dry weight/ activity  insulin-like -Mean pH value 100                                               ml μU/mg activity mU                      ______________________________________                                        Blood pH 7 1,2         11,0      13,2                                          pH 2,5 - 3,5                                                                            1,6         15,3      24,4                                          pH 9 - 10 1,3         18,6      24,1                                         Plasma pH 7                                                                              1,8          6,5      11,7                                          pH 2,5 - 3,5                                                                            2,7         14,7      39,7                                          pH 9 - 10 1,9          8,6      16,3                                         Erythrocyte                                                                   sediment (1:1)                                                                 pH 7      0,9          9,0       8,1                                          pH 2,5 - 3,5                                                                            2,8          5,3      14,8                                          pH 9 - 10 1,4         14,3      20,0                                         Pre-dialysed                                                                  blood                                                                          pH 7      0,3         16,0       5,0                                          pH 3      3,0         15,0      30,0                                          pH 9,5    3,5         30,0      90,0                                         ______________________________________                                    

EXAMPLE 1

Three liters of fresh blood taken from slaughter calves is mechanicallymixed with 300 ml of a 3.8% sodium citrate solution and 20 g of phenoland cooled to 4° C. After dilution with 900 ml of demineralised water,the pH is adjusted to 3.5 by the addition of about 100 ml of 6Nhydrochloric acid. The mixture is then warmed with stirring for 24 hoursto 35° to 40° C. Cooling to 4° C. is then again effected and the wholeis then dialysed in membrane tubes allowing for passage of molecules upto about 500 to 6000 Dalton against distilled water comprising 0.2%phenol. The ratio of inner to outer dialysate is about 1:5 to about1:10. The membrane tubes are mechanically agitated to optimise thedialysis effect. After 24 hours, the dialysate is renewed, and after afurther 24 hours this renewal is repeated. The inner dialysate may beworked up further in accordance with Example 4. The combined outerdialysates are concentrated down to 2 liters in a vacuum concentrator ata maximum temperature of 35° C. and the pH adjusted to 7.0 with 2Nammonia solution. The solution is then again concentrated to 2 litersvolume, possible precipitates centrifuged off, and the clear solutionfreeze-dried. One obtains 48 g of a substance mixture (dry) having aninsulin-like activity of 15.3 μU/mg.

EXAMPLE 2

The citrate-containing fresh calf blood (3 liters) described in Example1 is filled into centrifuge flasks up to 500 ml and centrifuged at 4° C.in a Christ centrifuge, model 4 KS, for 30 minutes at 3,400 revolutionsper minute. From 1 liter of blood, one obtains 0.6 to 0.75 liters ofremaining plasma, which is pipetted off and collected. The erythrocytesediment (0.30 to 0.45 liter) is diluted at a ratio of 1:1 with aqueous0.5% phenol and also collected for further treatment (see Example 3).One liter of the plasma portion is diluted with distilled watercontaining 0.5% phenol and the pH adjusted to 3.0 with 6N hydrochloricacid. The further heat treatment, dialysis, concentration andlyophilisation is carried out in a manner analogous to Example 1. Fromone liter of plasma, 27 g of dry substance having an insulin-likeactivity of 14.7 μU/mg is obtained.

EXAMPLE 3

The erythrocyte suspension obtained from 3 liters of calf blood inaccordance with Example 2 is adjusted to a pH of 9.0 with 5N ammoniasolution and treated with 200 to 300 ml ethanol to lower its viscosity.The erythrocytes Haemolise. After centrifuging, a clear solution isobtained. Heat treatment, and three dialyses, concentration andneutralisation with hydrochloric acid of the collected outer dialysatesin then carried out with following lyophilisation in accordance withExample 1. From 1 liter of 1:1 erythrocyte suspension 14 g of drysubstance is obtained with an insulin-like activity of 14.3 μU/mg.

EXAMPLE 4

The process described in Examples 1 to 3 is repeated, in which howeverthe inner dialysate obtained by dialysis of blood or a blood componentat neutral pH value is employed as starting material. For this purpose100 liters of a neutral inner dialysate (blood, plasma or erythrocytesuspension 1:1) is adjusted to a pH value of 3.5 with 6N hydrochloricacid while sirring. The resulting material is then treated with 100liters of 50% by volume ethanol to lower its viscosity, and this is thenincubated for 24 to 48 hours with stirring at 20° to 30° C., withprotection of 0.5% phenol solution. After cooling to 4° C. the 200liters of solution is again dialysed against a 0.2% aqueous phenolsolution. A strong swelling of the inner dialysate takes place which canbe kept within limits by stirring. The ratio of the volume of innerdialysate to outer dialysate lies between 1:2 to 1:5. After every 24hours, the outer dialysate is renewed. The collected outer dialysates(about 2,200 liters at a pH of 3.5) is concentrated down to 25 litersunder vacuum, neutralised with 5N ammonia solution, cloudiness filteredoff and further concentrated to 3.5 liters. The solution is then allowedto stand for one week at 4° C., precipitate filtered off and theremaining solution lyophilised after correction of the pH to value of6.9 to 7.0. From 100 liters of inner dialysate (corresponding to about85 liters of blood) one obtains about 350 g of dry substance having aninsulin-like activity of 15 to 20 μU/mg.

EXAMPLE 5

The process described in Example 4 is repeated, in which however thestarting material employed as inner dialysate for recoveringinsulin-like active substances is incubated and dialysed in the same wayat a pH of about 9.5. The outer dialysate thus obtained is thenneutralised with hydrochloric acid or acetic acid. In this process oneobtains from 100 liters of blood inner dialysate about 350 g of drysubstance having an insulin-like activity of 30 to 40 μU/mg.

EXAMPLE 6

Ten liters of freshly taken calf or swine blood is mechanically mixedwith 100 ml of a 3.8% sodium citrate solution and 60 g of phenol inaccordance with Example 1, cooled to 4° C. and diluted with 2 liters ofdemineralised water. The resulting solution is firstly clarified withthe aid of a centrifuge and then filtered through an ultrafiltrationmembrane (e.g. PSAC-Pellicon membranes of acetylcellulose) having apermeability up to 100 Dalton at 6 atmospheres pressure. In this manner,inorganic and organic components having a molecular weight of less thanabout 800 to 1200 Dalton are eliminated and a thick viscous pasteremains which is taken up in 2 liters of distilled water. The suspensionthus obtained is then treated in accordance with Example 1, 2 or 3 byacidifying or rendering alkaline and dialysing. The outer dialysateafter collection, neutralisation, concentration and lyophilising soobtained contains 25 to 30 g of dry substance having an insulin-likeactivity of 20 to 30 μU/mg.

EXAMPLE 7

4.5 g of the product obtained in accordance with Example 4 having aninsulin-like activity of 15 to 20 μU/mg is dissolved in 25 ml distilledwater. The solution is transferred to a column (50 × 100) containing astrongly cross-linked dextran gel having a fractionating range of up to700 Dalton (e.g. Sephadex 10, 40 to 120 μm cross-section). The column isthen eluted with 0.1N acetic acid at a rate of 60 ml/hour. Fractionseach of 12.4 ml are collected which are measured for UV absorption at206 and 254 millimicrons in a UV absorption photometer (e.g. UvicordIII), whereby three main peaks are obtained (fractions numbered 49 to79; 80 to 119 and 120 to 540). The main proportion of the inactive saltsare found in fraction II. The fraction I, with a yield of 115 mgcontains the main proportion of salt free substance having aninsulin-like activity of 60 μU/mg. The fraction III contains mainly lowmolecular weight inactive organic substances. The molecular weightdetermination of the substance (115 mg) from fraction I is carried outon a calibrated gel filtration column (26 × 100) containing cross-linkeddextran gel (e.g. Sephadex 25, medium 50 to 150 μm cross-section). Theelution with IN acetic acid (16 ml/hour) shows a main proportion ofabout 99% substance having a molecular weight of less than 1000 Dalton,the previous fraction (15 mg, i.e. 7.2% of the total organic content ofthe fraction) lies in the region between 350 and 5,000 Dalton and showsthe total insulin-like active activity of 150 μU/mg.

EXAMPLE 8

3 g of the product obtained according to Example 1 having aninsulin-like activity of 15.3 μU/mg dissolved in 15 ml of water aretransferred within 5 hours into a Hanish electrophoresis apparatus whichallows for fractionating into 90 fractions. The solution is diluted 1:1with chamber buffer which consists of a 1:3 diluted electrode buffer.The electrode buffer of a pH of 4.9 employed is a mixture of 100 mlpyridine and 80 ml glacial acetic acid which is diluted to 9000 ml withwater. The electrophoresis is carried out at a potential of 1800 to 1500volts and a current of from 150 to 165 mA at a temperature of 5° C. Thebuffer pump pumps 10 ml/hour and the dosing pump 1.5 ml/hour, and thus0.75 ml of the active solution. Nine fractions are separated, in whichthe fraction 6 (tubes 41 to 44), fraction 7 (tubes 45 to 48) andfraction 9 (tubes 53 to 55) show insulin-like activity. These fractionsare lyophilised. Fraction 6 provides 16 mg substance having aninsulin-like activity of 60 μU/mg, fraction 7 provides 12.8 mg having aninsulin-like activity of 150 μU/mg and fraction 9 provides 9.6 mg havingan insulin-like activity of 190 μU/mg. All three fractions show apositive ninhydrin and amido black G-reaction.

EXAMPLE 9

4 g of the product obtained in accordance with Example 3 having aninsulin-like activity of 14.3 μU/mg are dissolved in 100 ml of distilledwater, are transferred to a column (26 × 40) containg a cation exchangerbased on a polystyrene matrix (e.g. AG 50 W-x8) which is available inNH₄ -form and has a particle size of from 75 to 150 μm. After soaking inof the test solution, the column is washed with distilled water until nosubstance is shown to exist in the eluate (about 2 liters) by means ofan UV spectro-photometer (wave length 254 millimicrons). This eluate isdiscarded. The column is successively eluted with 200 ml of a 10%ammonia solution and 3 liters of distilled water. The collected eluateis concentrated under vacuum to one fifth of its original volume andthen freeze-dried. 550 mg of salt-free lyophilisate is thus obtainedwith an insulin-like activity of 32 μU/mg. An amino acid analysis of thefraction I obtained in Example 7 shows, before and after hydrolysis(under nitrogen with 6N hydrochloric acid at a temperature of 110° C.over a time period of 24 hours), the following values in percent:Ornithine und Lysine (12,23; 22,25), Histidine (1,30; 2,89), Arginine(traces; 0,47), Asparagine acid (0,71; 2,42), Threonine (0,61; 1,13),Serine (0.39; 1,85), Glutaminic acid (0,20; 2,16), Proline (1,38; 1,93),Glycine (0,15; 1,35), Alanine (1,54; 3,99), Cysteine (0,06; 0,08),Valine (1,12; 2,41), Methionine (0,17; 0,25), Isoleucine (0,14; 0,36),Leucine (0,16; 1,67), Tyrosine (0,52; 1,15) and Phenylalanine (Traces;0,10).

What we claim is:
 1. An organic peptide containing substance havinginsulin-like activity recovered from blood or a blood component of awarm-blooded animal, obtain by the steps of pretreating the blood orblood component by diluting the blood or blood component with from abouthalf to about four volumes of a hydrophilic diluent, adjusting the pH ofthe resulting solution to a value of from 2 to 4 or of from 8 to 10,incubating the pretreated blood at a temperature of from about 15° toabout 40° C. for a period of at least four hours, cooling the incubatedblood to a temperature of less than 10° C., dialysing the treated bloodafter neutralization against a membrane adapted to pass molecules havinga molecular weight of less than 6000, and recovering from dialysateorganic peptide substances having a molecular weight of from about 350to about 6000, and an insulin-like activity of from about 14 to about 40μU/mg solid.
 2. An organic peptide containing substance of claim 1recovered from blood or a blood component of a warm-blooded animal,having a molecular weight of from about 350 to 6,000, a non-suppressibleinsulin-like activity in the rat adipose tissue test of from about 35 to2000 μU/mg solids, solubility in ethanol, stability to heat at neutralpH, a positive ninhydrin and amido black G-reaction, an UV maximum at258 millimicrons indicating presence of purine derivatives, an aminoacid content after hydrolysis providing a peptide content of from 25 to30% of the dry weight of the salt free substance, with the followingamino acids: Glutamic acid, Leucine, Glycine, Serine, Asparginic acid,Alanine, Isoleucine, Histidine, Valine, Threonine, Lysine, Methionine,Tyrosine, Proline and Arginine, an insulin like activity indicated byuptake of glucose in adipose tissue, a blood sugar lowering effect inwarm-blooded animals, and an inhibition of lypolysis and the growthincrease of cell cultures.
 3. A peptide of claim 1 wherein the recoveredinsulin-like active substance is adjusted to obtain an isotonic solutionhaving a solids content of from about 2.5 to about 20 mg/ml, and ifnecessary to obtain the isotonic solution, adding glucose or sodiumchloride.
 4. A peptide of claim 1 wherein the recovered insulin-likeactive substance is adjusted to possess an insulin-like activity ofabout 500 μU/ml and, if necessary to obtain an isotonic solution, addingglucose or sodium chloride.